Application of Thermoplastic Microbioreactor to the Single Cell Study of Budding Yeast to Decipher the Effect of 5-Hydroxymethylfurfural on Growth

Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

Abstract:

Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.3299649

References:

[1] J. Hou, Z. Qui, H. Han, and Q. Zhang, “Toxicity evaluation of lignocellulose-derived phenolic inhibitors on Saccharomyces cerevisiae growth by using the QSTR method,” Chemosphere., vol. 201, pp. 286-293, Jun. 2018.
[2] A. Cavka, and L. J. Jöhnsson, “Comparison of the growth of filamentous fungi and yeasts in lignocellulose-derived media,” Biocatal. Agric. Biotechnol., vol. 3, no. 4, pp. 197-204, Oct. 2014.
[3] J. R. Almeida, A. Röder, T. Modig, B. Laadan, G. Liden, and M. Gorwa-Grauslund, “NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae,” Bioethanol Products and Process Engineering, vol. 78, no. 6, pp. 939-945, Apr. 2008.
[4] T. Hasunuma, K. S. K. Ismail, Y. Nambu, and A. Kondo, “Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural,” vol. 117, no. 2, pp. 165-169, Feb. 2014.
[5] Z. L. Liu, J. Moon, B. J. Andersh, P. J. Slininger, and S. Weber, “Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae,” Appl. Microbiol. Biotechnol., vol. 81, no. 4, pp. 743-753, Dec. 2008.
[6] M. Ma, and Z. L. Liu, “Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae,” BMC. Genomics., vol. 11, pp. 660, Nov. 2010.
[7] Z. L. Liu, M. Ma, and M. Song, “Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways,” vol. 282, no. 3, pp. 233-244, Sep. 2009.
[8] S. E. Park, H. M. Koo, Y. K. Park, S. M. Park, J. C. Park, and O. K. Lee, et. al., “Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae,” Bioresour. Technol., vol. 102, no. 10, pp. 6033-6038, May. 2011.
[9] N. T. Sehnem, S. Machado Ada, F. C. Leite, B. Pita Wde, M. A. Jr de Morais, and M. A. Ayub, “5-Hydroxymethylfurfural induces ADH7 and ARI1 expression in tolerant industrial Saccharomyces cerevisiae strain P6H9 during bioethanol production,” Bioresour. Technol., vol. 133, pp. 190-196, Apr. 2013.
[10] S. Puza, E. Gencturk, I. E. Odabasi, E. Iseri, S. Mutlu, and K. O. Ulgen, “Fabrication of cyclo olefin polymer microfluidic devices for trapping and culturing of yeast cells,” Biomed. Microdevices., vol. 19, no. 2, pp. 40, Jun. 2017.
[11] E. Gencturk, S. Mutlu, and K. O. Ulgen, “Advances in microfluidic devices made from thermoplastics used in cell biology and analyses,” Biomicrofluidics, vol. 11, no. 5, pp. 051502, Oct. 2017.
[12] T. A. Nissan, J. Bassler, E. Petfalski, D. Tollervey, and E. Hurt, “60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm,” EMBO J., vol. 21, no. 20, pp. 5539-5547, Oct. 2002.
[13] P. Mitchell, E. Petfalski, R. Houalla, A. Podtelejnikov, M. Mann, and D. Tollervey, “Rrp47p Is an Exosome-Associated Protein Required for the 3' Processing of Stable RNAs,” Mol. Cell. Biol., vol. 23, no. 19, pp. 6982-6992, Oct. 2003.
[14] D. L. Lafontaine, C. Bousquet-Antonelli, Y. Henry, M. Caizergues-Ferrer, and D. Tollervey, “The box H+ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase,” Genes. Dev., vol. 12, no. 4, pp. 527-537, Feb. 1998.
[15] N. S. Heiss, S. W. Knight, T. J. Vulliamy, S. M. Klauck, S. Wiemann, et. al., “X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions,” Nat. Genet., vol. 19, no. 1, pp. 32-38, May. 1998.
[16] A. K. Henras, R. Capeyrou, Y. Henry, and M. Caizergues-Ferrer, “Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs,” RNA., vol. 10, no. 11, pp. 1704-1712, Nov. 2004.
[17] X. Ma, C. Yang, A. Alexandrov, E. J. Grayhack, I. Behm-Ansmant, and Y. T. Yu, “Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism,” EMBO J., vol. 24, no. 13, pp. 2403-2413, Jul. 2005.
[18] T. M. Carlile, M. F. Rojas-Duran, B. Zinshteyn, H. Shin, K. M. Bartoli, and W. V. Gilbert, “Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells,” Nature, vol. 515, no. 7525, pp. 143-146, Nov. 2014.
[19] R. Rashid, B. Liang, D. L. Baker, O. A. Youssef, Y. He, K. Phipps, et. al., “Crystal Structure of a Cbf5-Nop10-Gar1 Complex and Implications in RNA-Guided Pseudouridylation and Dyskeratosis Congenita,” Mol. Cell., vol. 21, no. 2, pp. 249-260, Jan. 2006.
[20] J. Ge, and Y. T. Yu, “RNA pseudouridylation: new insights into an old modification,” Trends Biochem. Sci., vol. 38, no. 4, pp. 210-218, Apr. 2013.
[21] S. Schwartz, D. A. Bernstain, M. R. Mumbach, M. Jovanovic, R. H. Herbst, B. X. León-Ricardo, et. al., “Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA,” Cell., vol. 159, no. 1, pp. 148-162, Sep. 2014.
[22] C. Torchet, C. Jacq, and S. Hermann-Le Denmat, “Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae,” RNA., vol. 4, no. 12, pp. 1636-1652, Dec. 1998.
[23] H. R. Vos, R. Bax, A. W. Faber, J. C. Vos, and H. A. Raué, “U3 snoRNP and Rrp5p associate independently with Saccharomyces cerevisiae 35S pre-rRNA, but Rrp5p is essential for association of Rok1p,” Nucleic Acids Res., vol. 32, no. 19, pp. 5827-5833, Nov. 2004.
[24] S. Lebaron, A. Segerstolpe, S. L. French, T. Dudnakova, F. de Lima Alves, S. Granneman, et. al., “Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly,” Mol. Cell., vol. 52, no. 5, pp. 707-719, Dec. 2013.
[25] N. O. Bodnar, and T. A. Rapoport, “Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex,” Cell., vol. 169, no. 4, pp. 722-735, May. 2017.
[26] S. Böhm, D. Fisherman, and H. W. Mewes, “Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins,” Nucleic Acids Res., vol. 25, no. 12, pp. 2464-2469, Jun. 1997.
[27] M. E. Gardocki, M. Bakewell, D. Kamath, K. Robinson, K. Borovicka, and J. M. Lopes, “Genomic Analysis of PIS1 Gene Expression,” Eukaryot. Cell., vol. 4, no. 3, pp. 604-614, Mar. 2005.
[28] S. Rumpf, and S. Jentsch, “Functional Division of Substrate Processing Cofactors of the Ubiquitin-Selective Cdc48 Chaperone,” Mol. Cell., vol. 21, no. 2, pp. 261-269, Jan. 2006.
[29] C. Schuberth, H. Richly, S. Rumpf, and A. Buchberger, “Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation,” EMBO Rep., vol. 5, no. 8, pp. 818-824, Aug. 2004.
[30] R. Krick, S. Bremer, E. Welter, P. Scholetterhose, Y. Muehe, E. L. Eskelinen, et. al., “Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8,” J. Cell. Biol., vol. 190, no. 6, pp. 965-973, Sep. 2010.
[31] S. Böhm, and A. Buchberger, “The Budding Yeast Cdc48(Shp1) Complex Promotes Cell Cycle Progression by Positive Regulation of Protein,” PLoS One., vol. 8, no. 2, pp. e56486, Feb. 2013.
[32] M. Pantazopoulou, M. Boban, R. Foisner, and P. O. Ljungdahl, “Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation,” J. Cell. Sci., vol. 129. No. 20. Pp. 3770-3780, Oct. 2016.
[33] J. A. Stead, J. L. Costello, M. J. Livingstone, and P. Mitchell, “The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein,” Nucleic Acids Res., vol. 35, no. 16, pp. 5556-5567, Aug. 2007.
[34] H. Hieronymus, M. C. Yu, and P. A. Silver, “Genome-wide mRNA surveillance is coupled to mRNA export,” Genes. Dev., vol. 18, no. 21, pp. 2652-2662, Nov. 2004.
[35] S. Vodala, K. C. Abruzzi, and M. Rosbash, “The Nuclear Exosome and Adenylation Regulate Posttranscriptional Tethering of Yeast GAL Genes to the Nuclear Periphery,” Mol. Cell., vol. 31, no. 1, pp. 104-113, Jul. 2008.
[36] M. Feigenbutz, R. Jones, T. M. Besong, S. E. Harding, and P. Mitchell, “Assembly of the Yeast Exoribonuclease Rrp6 with Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47,” J. Biol. Chem., vol. 288, no. 22, pp. 15959-15970, May. 2013.
[37] E. Dedic, P. Seweryn, A. T. Jonstrup, R. K. Flygaard, N. U. Fedosova, S. V. Hoffman, et. al., “Structural analysis of the yeast exosome Rrp6p-Rrp47p complex by small-angle X-ray scattering,” Biochem. Biophys. Res. Commun., vol. 450, no. 1, pp. 634-640, Jul. 2014.
[38] M. Ask, M. Bettiga, V. Mapelli, and L. Olsson, “The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae,” Biotechnol. Biofuels., vol. 6, no. 1, pp. 22, Feb. 2013.
[39] J. Takeuchi, and A. Toh-e, “Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality,” Mol. Gen. Genet., vol. 262, no. 1, pp. 145-153, Aug. 1999.
[40] Y. Tone, N. Tanahashi, K. Tanaka, M. Fujimuro, H. Yokosawa, and A. Toh-e, “Nob1p, a new essential protein, associates with the 26S proteasome of growing Saccharomyces cerevisiae cells,” Gene., vol. 243, no. 1-2, pp. 34-45, Feb. 2000.
[41] J. M. Tkach, A. Yimit, A. Y. Lee, M. Riffle, M. Costanzo, D. Jaschob, et. al., “Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress,” Nat. Cell. Biol., vol. 14, no. 9, pp. 966-976, Sep. 2012.
[42] R. Egner, and K. Kuchler, “The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole,” FEBS Lett., vol. 378, no. 2, pp. 177-181, Jan. 1996.
[43] K. Miyahara, M. Mizunuma, D. Hirata, E. Tsuchiya, and T. Miyakawa, “The involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p and Snq2p in cation resistance,” FEBS Lett., vol. 399, no. 3, pp. 317-320, Dec. 1996.
[44] Y. Mahé, Y. Lemoine, and K. Kuchler, “The ATP Binding Cassette Transporters Pdr5 and Snq2 of Saccharomyces cerevisiae Can Mediate Transport of Steroids in Vivo,” J. Biol. Chem., vol. 271, no. 41, pp. 25167-25172, Oct. 1996.
[45] R. Emter, A. Heese-Peck, and A. Kralli, “ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms,” FEBS Lett., vol. 521, no. 1-3, pp. 57-61, Jun. 2002.
[46] Y. M. Mamnun, C. Schüller, and K. Kuchler, “Expression regulation of the yeast PDR5 ATP-binding cassette (ABC) transporter suggests a role in cellular detoxi¢cation during the exponential growth phase,” FEBS Lett., vol. 559, no. 1-3, pp. 111-117, Feb. 2004.
[47] A. L. Hitchcock, H. Krebber, S. Frietze, A. Lin, M. Latterich, and P. A. Silver, “The Conserved Npl4 Protein Complex Mediates Proteasome-dependent Membrane-bound Transcription Factor Activation,” vol. 12, no. 10, pp. 3226-3241, Oct. 2001.
[48] N. W. Bays, S. K. Wilhovsky, A. Goradia, K. Hodgkiss-Harlow, and R. Y. Hampton, “HRD4/NPL4 Is Required for the Proteasomal Processing of Ubiquitinated ER Proteins,” Mol. Biol. Cell., vol. 12, no. 12, pp. 4114-4128, Dec. 2001.